1) Adenylate cyclase activity is under the control of inhibitory and stimulatory agonists which act through specific surface receptors. The inhibitory receptors are coupled to the catalytic unit of cyclase through a guanine nucleotide-binding protein termed Gi. Specific binding of [3-H]guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was observed to membranes from cultured NG108-15 neuroblastoma x glioma hybrid cells; since inhibitory agonists induced release of Gpp(NH)p, it appeared that the binding was to Gi. Pertussis toxin, which catalyzes the ADP-ribosylation of Gi, reduced the inhibitory agonist-dependent release. To determine the mechanism for this effect, the binding of [3-H]Gpp(NH)p to ADP-ribosylated and unmodified transducin, a retinal protein similar to Gi, was examined. ADP-ribosylated transducin bound significantly less Gpp(NH)p than did the unmodified protein. Thus, the failure of agonist to release Gpp(NH)p from membranes from toxin-treated cells results from the inability of guanine nucleotide to bind. 2) NAD:arginine ADP-ribosyltransferases, which catalyze reactions similar to bacterial toxins, were previously identified in the cytosolic fraction of turkey erythrocytes. One such enzyme, termed transferase A, was activated by membrane components, such as lysophospholipids, as well as by nonionic and zwitterionic detergents; lysophospholipids are generated in response to activation of phospholipase A-2. This transferase modifies the arginine residues of proteins. In the case of ovine brain glutamine synthetase, ADP-ribosylation of one arginine resulted in loss of enzymatic activity; the ADP-ribosyltransferase thus recognizes specific arginine residues and may therefore have a role in regulating enzymatic function. In addition to the cytosolic ADP-ribosyltransferases A and B, two different transferases, termed C and A', were localized to the membrane and nuclear fractions, respectively. These transferases possess different physical, kinetic, and regulatory properties. The existence of a family of ADP-ribosyltransferases is consistent with a multifaceted role for mono-ADP-ribosylation in the cell.